HCV Real Time RT-PCR Kit User Manual For In Vitro
Contents
1. 9 Procedure 9 1 RNA Extraction Different brands of RNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For RNA extraction kit please comply with manufacturer s instructions The recommended extraction kit is as follows Nucleic Acid Isolation Kit Cat Number Manufacturer RNA Isolation Kit ME 0010 ZJ Biotech ME 0012 QIAamp Viral RNA Mini 52904 QIAGEN Extraction Kit 50 9 2 Internal Control It is necessary to add internal control IC in the reaction mix Internal control IC allows the user to determine and control the possibility of PCR inhibition among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink and smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and Transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 18ul 1 ul ul XPCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of ul IC Super Mix Enzyme Mix Internal Control io ee 1 The volumes of Super Mix and Enzyme Mix per reaction multiply wit2. O Revision No ZJ0002 Issue Date Jul 1 2012 iferiver HCV Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only IVD HR 0008 02 A B IVD For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 a Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument wal Shanghai ZJ Bio Tech Co Ltd z www liferiver com cn Tel 86 21 34680596 25 trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan road PuJiang Hi tech Park Shanghai China 1 Intended Use HCV real time RT PCR kit is used for the detection of HCV in serum or plasma by using real time PCR systems Its characteristics High sensitivity lower detection line 10 IU ml LOQ 210 1X 10 IU ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors Jf you use the RNA extraction kits recommended the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher High specificity test result will be positive only to hepatitis C virus Short operating time 2 and a half hours totally Good stability kept for 12 months at 20 C CV lt 5 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease as
3. Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains Super Mix for the specific amplification of HCV RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT HCV RNA is transcribed into cDNA Then a thermostable DNA polymerase is used to amplify the specific gene fragments by polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified HCV DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 In addition the kit can be used for identification of possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC Four quantitation standards are supplied allows the determination of the gene load 4 Kit Contents S Ref_ Cap Color Type of reagent Presentation 25rxns HCV Super Mix 1 vial 480ul RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control 1 vial 30ul Pink HCV QS1 5x10 TU ml 1 vial 20ul Purple HCV QS2 5x10 U ml 1 vial 20u1 Orange HCV QS3 5x10 IU ml 1 vial 20ul Yellow HCV QS4 5x10 IU ml 1 vial 20u1 QS Quantitation Standard e Biohazard waste container e Refrigerator and freezer 5 Storage e Sterile filter tips for micro pipets e Tube racks e All reagents should be stored at 20 C Storage at 4 C is not e Desktop microcentrifuge for eppendorf
4. e possible 1 The Ct value in channel FAM shows lt 38 The result is positive The sample contains HCV RNA Quantitative value of samples is automatically reported according to the standard curve Quantitative Value Data Analysis and Suggestion lt 10 IU ml HCV RNA Positive its concentration lower than 10 IU ml 10 2x10 IU ml HCV RNA Positive the quantitative value for recommendation only 2x10 10 IU ml HCV RNA Positive the quantitative value is valid gt 10 IU ml 1 HCV RNA Positive but the quantitative value for recommendation only 2 Re test the sample after dilute the sample by several times making the quantitative value within 2 X 10 10 IU ml 2 The Ct value in channel FAM shows 38 40 please repeat again If the result still shows 38 40 it can be considered negative 3 In channel FAM no signal is detected at the same time a HEX VIC JOE signal from the Internal Control appears The sample does not contain any HCV RNA It can be considered negative 4 Neither in channel FAM nor in channel HEX VIC JOE is a signal detected A diagnostic statement can not be made Inhibition of the RT PCR reaction For further questions or problems please contact our technical support at trade liferiver com cn
5. h the number of samples which includes the number of controls standards and sample 5ul 20ul prepared Molecular Grade Water is used as the negative control For reasons of Extraction RNA Master Mix unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge rae 2 Pipet 20u Master Mix with micropipets in sterile filter tips to each Real time PCR reaction plate tubes Separately add 5ul RNA sample QS1 QS2 QS3 QS4 and Reaction negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination Plate Tube 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes PCR Instrument 4 Perform the following protocol in the instrument 5 AIF you use ABI Prism system please choose none as passive reference and quencher 45 C for 10 min 1 cycle 95 C for 15 min 1 cycle 95 C for 15 sec 60 C for 60 sec 40 cycles Fluorescence is measured at 60 C channel FAM and HEX VIC JOE should be chosen 10 Threshold setting just above the maximum level of molecular grade water 11 Calabration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control Channel Control FAM Target Nucleic Acid HEX VIC JOE IC Molecular Grade Water _ 25 35 13 Data Analysis and Interpretation The following results ar
6. say During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description HCV is an enveloped RNA virus in the flaviviridae family which appears to have a narrow host range It is a major cause of acute hepatitis and chronic liver disease including cirrhosis and liver cancer Globally an estimated 170 million persons are chronically infected with HCV and 3 to 4 million persons are newly infected each year Twenty percent of persistently infected individuals will develop liver cirrhosis and hepatocellular carcinoma occurs in up to 2 5 HCV is spread primarily by direct contact with human blood The major causes of HCV infection worldwide are use of unscreened blood transfusions and re use of needles and syringes that have not been adequately sterilized HCV real time RT PCR kit contains a specific ready to use system for HCV detection for genotype IV by Reverse
7. type tubes RCF max recommended 16 000 x g e All reagents can be used until the expiration date indicated on the kit label 7 Warnings and Precaution e Repeated thawing and freezing gt 3x should be avoided as this e Carefully read this instruction before starting the procedure may reduce the sensitivity of the assay e For in vitro diagnostic use only e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Real time PCR reaction tubes plates Vortex mixer e Pipets 0 5 ul 1000 ul e Cryo container e Disposable gloves powderless e Sterile microtubes e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate
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